Silver nanoparticle enhanced chemiluminescence method for the determination of nitrazepam.

We report on a simple and sensitive chemiluminescence (CL) method to determine nitrazepam. This method is based on the fact that rhodamine 6G (Rh6G) enhanced the weak CL emission of the reaction of hexacyanoferrate with nitrazepam, and that it was further enhanced by silver nanoparticles (AgNPs). The effects of the concentrations of K3Fe(CN)6, Rh6G, AgNPs and NaOH on the CL reaction were investigated. Under the optimum conditions, the CL intensity was proportional to the concentration of nitrazepam in the range from 1.0 nM to 10.0 µM. The detection limit (3σ) was at 0.1 nM. The relative standard deviation was 2.1% (at a 0.1 µM concentration and for n = 11). The method was successfully applied to the determination of nitrazepam in Coca-Cola beverage, urine and plasma, and the recovery was 98 - 103%. We also considered the possible CL reaction mechanism.

Simultaneous quantification of urine flunitrazepam, nimetazepam and nitrazepam by using liquid chromatography tandem mass spectrometry.

BACKGROUND: Benzodiazepines are used in hypnotics, sedation, and anti-anxiety. Recently liquid chromatography tandem mass spectrometry (LC-MS/MS) has been vastly developed for drug analysis in biological samples. METHODS: We developed and validated a LC-MS/MS method for simultaneous quantification of flunitrazepam (FM2), nimetazepam and nitrazepam levels in 87 benzodiazepine positive human urine specimens by enzyme immunoassay. Deuterated analogues were used as internal standard. RESULTS: The limits of quantification were found to be 0.25, 2.5, 5, 5 and 1ng/ml for FM2, 7-aminoFM2, nimetazepam, 7-amino-nimetazepam and nitrazepam, respectively. The intraday and inter-day CVs ranged from 0.6 to 4.6% and 1.2-9.4%, respectively. The within-day accuracy ranged from 80.8 to 108.7% and the between-day accuracy ranged from 80.5 to 118.0%. The recovery rate ranged from 70.5 to 96.7% for five different analytes. A group of 34 urine samples previously gas chromatography-mass spectrometry determined to contain 7-aminoFM2 was analyzed by this new LC-MS/MS approach. Quantitative data produced by both methods agreed well. CONCLUSIONS: The LC-MS/MS method has proved to be robust and specific for the quantification of FM2, nimetazepam and nitrazepam in urine samples. This study also confirmed that nitrazepam and 7-aminonimetazepam are the metabolic products of nimetazepam.

Determination of nimetazepam and 7-aminonimetazepam in human urine by using liquid chromatography-tandem mass spectrometry.

We report determination of metabolites of popular drugs of abuse, including nimetazepam and nitrazepam, in urine by using liquid chromatography/mass spectrometry. Nimetazepam and its metabolites, 7-aminonimetazepam and nitrazepam, were extracted by solid-phase extraction using a DAU cartridge. An ammonium acetate buffer solution (pH 4) and a Luna polar-RP column were selected as the mobile and stationary phase, respectively, for liquid chromatography. Mass spectrometry was used for analysis and was optimized for operation in the positive mode for all analytes. The urine specimens were screened for the presence of nimetazepam and its metabolites nitrazepam and 7-aminonimetazepam at a concentration of 0.1ng/mL. Presence of 7-aminonimetazepam in the urine was an indicator of the subject being a probable abuser of nimetazepam.

Measurement of benzodiazepines in urine by liquid chromatography-tandem mass spectrometry: confirmation of samples screened by immunoassay.

BACKGROUND: Liquid chromatography linked to tandem mass spectrometry (LC/MS/MS) provides the ability to identify a range of benzodiazepines in accordance with European Union criteria and is an attractive method for the confirmation of benzodiazepines following immunoassay screening. METHODS: An LC/MS/MS method to detect and quantitate the six most common benzodiazepines/metabolites (diazepam, nitrazepam, nordiazepam, oxazepam, temazepam and 7-aminonitrazepam) was developed together with a qualitative screening method for a further 11 benzodiazepines/metabolites. These methods were used for confirmation of 250 urine samples submitted for routine drug screening by immunoassay for benzodiazepines (100 samples positive for a benzodiazepine, assay cut-off >200 microsg/L). RESULTS: The lower limits of detection and quantitation were less than 2.5 and 5 microg/L for the six most common benzodiazepines. Recoveries ranged between 97% and 102% and calibration curves were linear to at least 4000 microg/L (r = 0.99). Intra and inter-assay imprecision were <10% (n = 10) and <20% (n = 15), respectively. Confirmation of benzodiazepines using LC/MS/MS was achieved for 89% of the immunoassay-positive urine samples. Of the immunoassay-negative urine samples, 31% of these demonstrated a benzodiazepine using LC/MS/MS. CONCLUSION: The validated LC/MS/MS method developed is effective for the confirmation of immunoassay screening results for benzodiazepines. The lower limit of detection and assay specificity offers a longer window of detection and more detailed clinical information compared with immunoassay screening.

A highly sensitive method for the determination of 7-aminonitrazepam, a metabolite of nitrazepam, in human urine using high-performance electrospray liquid chromatography tandem mass spectrometry.

A highly sensitive method has been developed for the determination of urinary 7-aminonitrazepam (7-ANZP), the major metabolite of nitrazepam, using high-performance electrospray liquid chromatography tandem mass spectrometry. The samples were prepared for analysis by adding 7-aminoclonazepam (7-ACZP, internal standard), hydrolysis with beta-glucuronidase and liquid-liquid extraction. Mass spectral acquisition was achieved by selectively monitoring the reaction between the two diagnostic transition reactions. Qualitative analysis was based on the retention time, and the quantitation was carried out by comparison with the internal standard. The recoveries of different concentrations of 7-ANZP from spiked blank samples was 89.0-95.2%, and the relative standard deviation was below 6.4%. The limit of determination in urine was 0.07 ng/mL, and the limit of quantitation was 0.5 ng/mL in the linear range of 1-50 ng/mL. This method possesses the merits of convenient operation, high sensitivity and good repeatability, making it a practical method for analysis of 7-ANZP in urine.

Long-term stability of various drugs and metabolites in urine, and preventive measures against their decomposition with special attention to filtration sterilization.

The long-term stability of drugs and metabolites of forensic interest in urine, and preventive measures against their decomposition have been investigated, with special attention to filtration sterilization. An aseptic urine collection kit, which was recently developed based on filtration sterilization, was utilized for the aseptic collection and storage of urine samples. For evaluating preservation measures, methamphetamine (MA), amphetamine (AP), nitrazepam (NZ), estazolam (EZ), 7-aminoflunitrazepam (7AF), cocaine (COC), and 6-acetylmorphine (6AM) were spiked into urine at 500 ng/mL each, and were monitored for 6 months at 25, 4, and -20 degrees C, after the addition of NaN(3) and/or filtration sterilization using the aseptic collection kit. In severely contaminated urine with bacteria, there were significant losses of 7AF and NZ, and slight decomposition of MA and AP at 25 degrees C. However, such degradation was successfully suppressed by the use of the kit, though the use of the kit and NaN(3) were preferred for 7AF. The kit was also effective in preventing the hydrolyses of COC and 6AM, while it was suggested that the common preservative NaN(3) can accelerate the hydrolysis of such ester-type drugs and metabolites.

Extraction and analysis of flunitrazepam/7-aminoflunitrazepam in blood and urine by LC-PDA and GC-MS using butyl SPE columns.

The forensic toxicology community has recognized flunitrazepam and its metabolite (7-aminoflunitrazepam) as compounds of concern for several years. In this procedure, the analytes were extracted from whole blood and urine onto single mode solid phase cartridges (butyl) using nitrazepam as an internal standard. The columns were washed with distilled water and hexane. All three compounds were eluted from the sorbent using an ethyl acetate-methanol solvent mixture. After collection and evaporation of the solvent, the residue was dissolved in A, 0.1% (v/v) aqueous trifluoroacetic acid for HPLC-PDA analysis or B, ethyl acetate for derivatization with pentafluoropropionic anhydride (PFPA) for analysis by gas chromatography-mass spectrometry (selected ion monitoring, SIM). A limit of quantitation for this method using HPLC-PDA was found to be 5 and 1.0 ng mL(-1) by SIM.

[Analysis of benzodiazepine derivative mixture by gas-liquid chromatography]. / Benzodiazepino dariniu misinio analize duju ir skysciu chromatografijos metodu.

The analysis of mixture of benzodiazepine derivates (chlordiazepoxide, flunitrazepam, medazepam, nitrazepam, oxazepam and tetrazepam) by gas--liquid chromatography (GLC) in purpose to separate and identify these psychotropic drugs in mixture is presented in this article. The experiment was carried out in vitro, accommodating this method for identification and separation of drugs, isolated from biological objects (blood and urine). Referring to data of annual reports of chemical investigations (1) above-mentioned psychotropic drugs are very frequent among drug intoxication. In most cases they are detected in the mixture of the same or different pharmacological group, and this causes difficulty for separation and identification. The analysis of the mixture was carried out by GLC, which is widely used in practice of forensic-chemical examination. Adsorbents and stationery phases were changed; the conditions and parameters of chromatography were modified, in purpose totally separate preparations in the mixture. For the separation and identification of all three preparation the column packed with Inerton Super with stationary phase 3% OV-17 is suitable. The column temperature-290 degrees C. The mixture of these drugs was excreted from body fluids (blood and urine) in vitro and investigated by GLC under these conditions. The results of investigation were similar.

[Analysis of 7-aminonitrazepam in urine by trimethylsilyl derivatization-gas chromatography/mass spectrometry].

A highly sensitive method has been developed for the analysis of 7-aminonitrazepam (7-ANIZ), the major metabolite of nitrazepam, in urine by trimethylsilyl derivatization-gas chromatography/mass spectrometry. Urine samples were extracted with ethyl ether-ethyl acetate (99:1, volume ratio). The extracts were derivatized with N,O-bis (trimethylsilyl) trifluoroacetamide, and the total ion current chromatograms of derivatives were acquired. 7-ANIZ was identified by the relative abundance of major characteristic ions in the mass spectrum of its derivative and the retention time of the mass chromatogram peaks of these characteristic ions. Based on the mass chromatogram of the base peak ion, quantification was performed using 7-aminoclonazepam (7-ACLZ) as the internal standard. The extraction efficiency of 7-ANIZ was 82.8%. The linear range was 10 microg/L - 500 microg/L. The limit of detection was 1.2 microg/L and the limit of quantification was 3.5 microg/L. The recoveries were 94.7% - 103.5%, and the RSDs were 3.9% - 5.4%. 7-ANIZ in the urine sample excreted by the subject over 96 h period after oral administration of 10 mg nitrazepam was measured. It is demonstrated that the method can be applied to the forensic identification.

Postmortem distribution and redistribution of nitrobenzodiazepines in man.

The distribution of the nitrobenzodiazepines, flunitrazepam, clonazepam and nitrazepam, and their respective 7-amino metabolites were examined in blood, serum, vitreous humor, liver, bile and urine of decedents taking these drugs. Peripheral blood, serum and liver concentrations were not significantly different to each other. However, vitreous concentrations were one-third of blood, while bile concentrations were 5-12 fold higher. Blood, serum and vitreous contained predominantly the 7-amino metabolite, liver contained only the metabolite, while bile contained significant concentrations of both the parent drug and the 7-amino metabolite. Urine contained only small concentrations of parent drug, however, as expected a number of metabolites were detected. Redistribution studies compared the drug concentrations of femoral blood, taken at body admission to the mortuary, with femoral blood taken at autopsy approximately 39 h later in 48 cases. The concentrations of 7-amino metabolites were not significantly different, however the concentrations of parent nitrobenzodiazepines were significantly higher in the admission specimens. In 6 cases in which subclavian blood was taken, the concentrations were not significantly different to the concentrations in admission blood. Similar findings were observed when femoral and subclavian blood concentrations were compared in 6 cases. There was also no apparent difference in total blood concentrations of nitrobenzodiazepines when blood concentrations taken in hospital shortly prior to death were compared to postmortem blood. Postmortem diffusion into peripheral blood is therefore not a confounding factor in the interpretation of nitrobenzodiazepine concentrations.

Benzodiazepine misuse by drug addicts.

Using a high-performance liquid chromatography method, we measured seven commonly prescribed benzodiazepines (chlordiazepoxide, nitrazepam, nordiazepam, oxazepam, lorazepam, temazepam and diazepam) in 100 urine samples obtained from patients attending the Leeds Addiction Unit. All of the urines selected for investigation were positive for benzodiazepines using an EMIT (Enzyme Immunoassay) screen. Forty-four of the urines contained a range of benzodiazepines, none of which had been prescribed. Nitrazepam was detected most frequently (61 urine samples), but had not been prescribed to any of the patients in this study. Chlordiazepoxide was detected in 49 urine samples, although it had been prescribed to only five patients. Temazepam was detected in 28 urine samples. Fourteen patients providing 21 urine samples had been prescribed temazepam for treatment. However, temazepam was detected in only 14 of these samples. Multiple benzodiazepine abuse was evident from the high rate of detection of unrelated benzodiazepines.

Simultaneous determination of nitrazepam and its metabolites in urine by micellar electrokinetic capillary chromatography.

We applied micellar electrokinetic capillary chromatography to simultaneous separation and determination of nitrazepam and its major metabolites, 7-aminonitrazepam and 7-acetamidonitrazepam, in spiked urine. Prior to electrophoresis, the three compounds were successfully extracted from the spiked urine with commercial disposable solid-phase cartridges. The optimum running buffer for the separation was prepared by combining 85 parts of 60 mM sodium dodecyl sulphate-6 mM phosphate-borate, adjusted to pH 8.5, with 15 parts of methanol. The separation order, completed within 25 min, was 7-aminonitrazepam > 7-acetamidonitrazepam > nitrazepam, at an applied potential of 20 kV. We obtained reproducible electropherograms in successive repetitions, and few other peaks or interferences appeared in the electropherogram. The detection limits of the three compounds were 50-100 pg (0.1-0.2 microgram/ml of analyte in spiked urine), and the recoveries were 78.9-100.8% for 1 microgram/ml and 84.1-100.3% for 5 micrograms/ml. The application of this method to forensic or clinical samples is demonstrated.

Application of Empore C-8 extraction disks for screening urine in systematic toxicological analysis.

Solid-phase extraction (SPE) by means of disposable columns has become a widely accepted technique for sample pretreatment in toxicology, both for directed analyses and for screening analyses. However, the sample capacity in SPE is usually limited to a few millilitres. Therefore, we have investigated to what extent these problems can be overcome by using Empore extraction disks, consisting of chemically modified C-8 reversed-phase silica, embedded in an inert polytetrafluoroethylene (PTFE) matrix. Human urine was selected as the matrix and dexetimide and mepyramine were initially used as test drugs because these drugs were available in tritiated form. Additional drugs investigated included codeine, hexobarbital, imipramine, methamphetamine, and nitrazepam. In these investigations, the sample capacity for untreated urine was at least 25 mL, and analyte quantities up to 250 micrograms could be retained by these filters. Washing with water/methanol mixtures was successful in removing substantial amounts of endogenous interferences, and methanol proved to be an acceptable eluent. Thus, these disks seem to have interesting potential for toxicological analysis in that sample concentration and cleanup can be achieved at the same time.

Detection of benzodiazepine intake in therapeutic doses by immunoanalysis of urine: two techniques evaluated and modified for improved performance.

We evaluated the EMIT (enzyme-multiplied immuno technique) and FPIA (fluorescence polarization immunoassay) urine screening systems for detection of benzodiazepine intake. Healthy male volunteers were given single oral therapeutic doses of alprazolam (2 mg), chlordiazepoxide (25 mg), flunitrazepam (1 mg), lorazepam (3.75 mg), nitrazepam (5 mg), and triazolam (0.25 mg), after which urine was collected for the next 32 h. The EMIT method failed to detect the intake of flunitrazepam, lorazepam, and nitrazepam. FPIA did not detect the intake of chlordiazepoxide, flunitrazepam, lorazepam, nitrazepam, and triazolam. Modification of the EMIT method to include enzymatic hydrolysis did not significantly alter the results obtained with this method. A modification of the FPIA method to include enzymatic hydrolysis and a lower cutoff value improved the results considerably, so that we reliably detected all studied substances but flunitrazepam. We conclude that (a) both EMIT and FPIA techniques, when used as intended by the manufacturers, are unreliable for the detection of intake of therapeutic doses of these benzodiazepines, and (b) the described modification of the FPIA should provide a much improved tool for detection of benzodiazepine intake.

Involvement of the intestinal microflora in nitrazepam-induced teratogenicity in rats and its relationship to nitroreduction.

A study was undertaken to investigate the relationship between nitroreduction of nitrazepam and its teratogenic effects and the involvement of the intestinal microflora in Sprague-Dawley rats. Incubation of bacterial suspensions from rat cecal contents with nitrazepam resulted in extensive reduction to 7-aminonitrazepam. Rat liver homogenates also reduced nitrazepam but only under anaerobic conditions. Following oral administration of 300 mg/kg nitrazepam to pregnant rats, total excretion of reduced metabolites (7-aminonitrazepam and 7-acetylaminonitrazepam) in urine and feces accounted for approximately 30% of the administered dose. When antibiotics were administered to dams to deplete their intestinal microflora prior to administration to nitrazepam, the total excretion of the reduced metabolites in the urine and feces decreased to 2% of the dose. Nitroreductase activity of cecal contents was almost completely suppressed by antibiotic pretreatment, but the activity of liver homogenates was not significantly altered by the same treatment. The incidence of nitrazepam-induced malformations was markedly decreased by antibiotic pretreatment. These results suggest that the intestinal microflora plays an important role in the reductive metabolism of nitrazepam and that the teratogenicity of nitrazepam may be related to its nitroreduction by the microflora.

Direct derivative spectrophotometric determination of nitrazepam and clonazepam in biological fluids.

The use of fifth order derivative spectra allows the direct determination of nitrazepam in urine at 388 nm with a limit of detection of 1 microgram ml-1. The determination of nitrazepam in blood plasma can be carried out directly by measurement at 402 nm in the fourth order derivative spectra with a limit of detection of 1.5 micrograms ml-1. Clonazepam can be determined directly in urine samples at 384 nm by using sixth order derivative spectra with a limit of detection of 1 microgram ml-1 and in blood plasma by using fourth order derivative spectra at 384 nm with a limit of detection of 0.5 ppm.

Determination of nitrazepam and its main metabolites in urine by thin-layer chromatography and direct densitometry.

A method for the direct quantitative densitometry of nitrazepam and its main metabolites (7-aminonitrazepam, 7-acetamidonitrazepam and 2-amino-5-nitrobenzophenone) in urine was developed. The unchanged drug and its metabolites were extracted with benzene-dichloromethane (4:1), subjected to thin-layer chromatography, and determined by direct ultraviolet densitometry. Recovery experiments showed that the method was quantitative. The limit of detection was 5 ng/ml for 2-amino-5-nitrobenzophenone and 10 ng/ml for other compounds. The method was applied to the determination of nitrazepam and its metabolites excreted in human urine after administration of 10 mg of the drug.